This application is a 371 of PCT/EP 99/04287 filed Jun. 21, 1999.
The present invention relates to epothilone analogs having side chain modifications and to methods for producing such compounds, their use in the therapy of diseases or for the manufacture of pharmaceutical preparations for the treatment of diseases, as well as to novel intermediates used in the synthesis of such analogs and new methods of synthesis.
The epothilones (1-5) are natural substances which exhibit cytotoxicity even against paclitaxel-resistant tumor cells by promoting the polymerization of xcex1- and xcex2-tubulin subunits and stabilizing the resulting microtubule assemblies. Epothilones displace paclitaxel (the active principle of TAXOL(trademark)) from its microtubuli binding site and are reported to be more potent than paclitaxel with respect to the stabilization of microtubules. 
What is needed are analogs of epothilone A and B that exhibit superior pharmacological properties, especially one or more of the following properties: an enhanced therapeutic index (e.g. a larger range of cytotoxic doses against e.g. proliferative diseases without toxicity to normal cells), better pharmakokinetic properties, better pharmacodynamic properties, better solubility in water, better efficiency against tumor types that are or become resistant to treatment with one or more other chemotherapeutics, better properties to facilitate manufacture of formulations, e.g. better solubility in polar solvents, especially those comprising water, enhanced stability, convenient manufacture of the compounds as such, improved inhibition of proliferation at the cellular level, high levels of microtubule stabilizing effects, and/or specific pharmacologic profiles.
The present invention relates to new compounds that surprisingly have one or more of the above-mentioned advantages.
One major aspect of the invention relates to an epothilone analog compound represented by the formula I 
wherein
the waved bond indicates that bond xe2x80x9caxe2x80x9d is present either in the cis or in the trans form;
(i) R2 is absent or oxygen; xe2x80x9caxe2x80x9d can be either a single or double bond; xe2x80x9cbxe2x80x9d can be either absent or a single bond; and xe2x80x9ccxe2x80x9d can be either absent or a single bond, with the proviso that if R2 is oxygen then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are both a single bond and xe2x80x9caxe2x80x9d is a single bond; if R2 is absent then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent and xe2x80x9caxe2x80x9d is a double bond; and if xe2x80x9caxe2x80x9d is a double bond, then R2, xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent;
R3is a radical selected from the group consisting of hydrogen; lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; and xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3;
R4 and R5 are independently selected from hydrogen, methyl or a protecting group, preferably hydrogen; and
R1 is a radical selected from the following structures: 
xe2x80x83aspect of the invention, of the formula 
wherein R and Rxe2x80x2 are lower alkyl, especially methyl, or, in a broader aspect of the invention, furthermore Rxe2x80x2 is hydroxymethyl or fluoromethyl and R is hydrogen or methyl;
(ii) and, if R3 is lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; or xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3, and the other symbols except R1 have the meanings given above, R1 can also be a radical selected from the following structures: 
xe2x80x83or, if R3 has one of the meanings given in the definition of R3 above under (ii) other than methyl, R1 can also be a radical of the formula 
(iii) and, if R3 is hydrogen, lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; or xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3, and
R2 is oxygen, xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are each a single bond and xe2x80x9caxe2x80x9d is a single bond, then R1 can also be a radical of the partial formula: 
(iv) and, if R3 is lower alkyl other than methyl, especially ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; or preferably is xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; or xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3; and the other symbols except for R1 have the meanings given above under (i), R1 can also be a moiety of the formula 
or a salt of a compound of the formula I where a salt-forming group is present.
A further aspect of the invention relates to a method of synthesis of a compound of the formula 
(wherein Q is hydrogen or preferably methyl) and/or a method of synthesis of a compound of the formula 
The general terms used hereinbefore and hereinafter preferably have within the context of this disclosure the following meanings, unless otherwise indicated:
The term xe2x80x9clowerxe2x80x9d means that the respective radical preferably has up to and including 7, more preferably up to and including 4 carbon atoms.
Lower alkyl can be linear or branched one or more times and has preferably up to and including 7, more preferably up to and including 4 carbon atoms. Preferably, lower alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl or further n-pentyl or n-hexyl.
A protecting group is preferably a standard protecting group. If one or more other functional groups, for example carboxy, hydroxy, amino, or mercapto, are or need to be protected in a compound of formulae I, because they should not take part in the reaction, these are such groups as are usually used in the synthesis of peptide compounds, and also of cephalo-sporins and penicillins, as well as nucleic acid derivatives and sugars.
The protecting groups may already be present in precursors and should protect the functional groups concerned against unwanted secondary reactions, such as acylations, etherifications, esterifications, oxidations, solvolysis, and similar reactions. It is a characteristic of protecting groups that they lend themselves readily, i.e. without undesired secondary reactions, to removal, typically by solvolysis, reduction, photolysis or also by enzyme activity, for example under conditions analogous to physiological conditions, and that they are not present in the end-products. The specialist knows, or can easily establish, which protecting groups are suitable with the reactions mentioned hereinabove and hereinafter.
The protection of such functional groups by such protecting groups, the protecting groups themselves, and their removal reactions are described for example in standard reference works, such as J. F. W. McOmie, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, London and New York 1973, in T. W. Greene, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, Wiley, New York 1981, in xe2x80x9cThe Peptidesxe2x80x9d; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in xe2x80x9cMethoden der organischen Chemiexe2x80x9d (Methods of organic chemistry), Houben Weyl, 4th edition, Volume 15/I, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, xe2x80x9cAminosxc3xa4uren, Peptide, Proteinexe2x80x9d (Amino acids, peptides, proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in Jochen Lehmann, xe2x80x9cChemie der Kohlenhydrate: Monosaccharide und Derivatexe2x80x9d (Chemistry of carbohydrates: monosaccharides and derivatives), Georg Thieme Verlag, Stuttgart 1974. Especially preferred protecting groups are hydroxy protecting groups, such as tert-butyldimethylsilyl or trityl.
R4 and R5 are preferably hydrogen.
The waved bond starting from the carbon atom bearing R3 means that bond xe2x80x9caxe2x80x9d is present in the trans- or preferably the cis-form.
Salts are especially the pharmaceutically acceptable salts of compounds of formula I.
Such salts are formed, for example, as acid addition salts, preferably with organic or inorganic acids, from compounds of formula I with a basic nitrogen atom, especially the pharmaceutically acceptable salts. Suitable inorganic acids are, for example, halogen acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulfonic or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, maleic acid, tartaric acid, citric acid, amino acids, such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid, dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamic acid, or other organic protonic acids, such as ascorbic acid.
For isolation or purification purposes it is also possible to use pharmaceutically unacceptable salts, for example picrates or perchlorates. For therapeutic use, only pharmaceutically acceptable salts or free compounds are employed (where applicable in the form of pharmaceutical preparations), and these are therefore preferred.
In view of the close relationship between the novel compounds in free form and those in the form of their salts, including those salts that can be used as intermediates, for example in the purification or identification of the novel compounds, any reference to the free compounds hereinbefore and hereinafter is to be understood as referring also to the corresponding salts, as appropriate and expedient.
The term xe2x80x9caboutxe2x80x9d in connection with numerical values, e.g. xe2x80x9cabout 2-fold molar excessxe2x80x9d or the like, is preferably intended to mean that the given numerical value may deviate from the given number by up to xc2x110%, more preferably by up to xc2x13%; most preferably, the numerical value is exactly as given.
In a preferred embodiment of the invention, the compounds of formula I as described under (iv) above 
are excluded from the scope of the invention.
Also, a group of compounds of the formula I without a compound of the formulae I wherein R1 is a moiety of any one of the formulae 
is preferred (the remaining symbols having the meanings defined for a compound of the formula I).
Especially preferred is either a free compound of the formula I, or a salt thereof.
Bioactivity: The compound(s) of the invention can be used for the treatment of a proliferative disease, especially a cancer, like cancers of the lung, especially non-small lung cell lung carcinoma, of the prostate, of the intestine, e.g. colorectal cancers, epidermoid tumors, such as head and/or neck tumors, or breast cancer, or other cancers like cancers of the bladder, pancreas or brain or melanoma, including especially the treatment of cancers that are multidrug-resistant (e.g. due to the expression of p-glycoprotein=P-gp) and/or refractory to treatment with paclitaxel (e.g. in the form of TAXOL).
Biological Evaluation
The ability of the compounds of the present invention to block the depolymerization of microtubuli can be shown by the following assay:
Microtubule assays are carried out following literature procedures and evaluate synthesized compounds for their ability to form and stabilize microtubules. Cytotoxicity studies are carried out as well.
The compounds of formula I are tested for their action on tubulin assembly using purified tubulin with an assay developed to amplify differences between compounds more active than Taxol. Compounds of the formula I are found to have a high level of cytotoxic and tubulin polymerization activity, as compared to Epothilones A and B. (Lin et al. Cancer Chemother. Pharmacol. 38, 136-140 (1996); Rogan et al. Science 244, 994-996 (1984)).
Filtration Colorimetric Assay
Microtubule protein (0.25 ml of 1 mg/ml) is placed into an assay tube and 2.5 xcexcl of the test compound are added. The sample is mixed and incubated at 37xc2x0 C. for 30 min. Sample (150 xcexcl) is transferred to a well in a 96-well Millipore Multiscreen Durapore hydrophilic 0.22 xcexcm pore size filtration plate which has previously been washed with 200 xcexcl of MEM buffer under vacuum. The well is then washed with 200 xcexcl of MEM buffer. To stain the trapped protein on the plate, 50 xcexcl amido black solution [0.1% naphthol blue black (Sigma)/45% methanol/10% acetic acid] are added to the filter for 2 min; then the vacuum is reapplied. Two additions of 200 xcexcl amido black destain solution (90% methanol/2% acetic acid) are added to remove unbound dye. The signal is quantitated by the method of Schaffner and Weissmann et al. Anal. Biochem., 56: 502-514, 1973 as follows: 200 xcexcl of elution solution (25 mM NaOH-0.05 mm EDTA-50% ethanol) are added to the well and the solution is mixed with a pipette after 5 min. Following a 10-min incubation at room temperature, 150 xcexcl of the elution solution are transferred to the well of a 96-well plate and the absorbance is measured on a Molecular Devices Microplate Reader.
Cytotoxicity experiments with 1A9, 1A9PTX10 (xcex1-tubulin mutant), and 1A9PTX22 (xcex1-tubulin mutant) cell lines can reveal the cytotoxic activity of the compounds of formula I. Like the naturally occurring epothilones 1 and 2, compounds of the formula I show significant activity against the altered xcex1-tubulin-expressing cell lines 1A9PTX10 and 1A9PTX22. For compounds of the formula I, the preferred IC50 values (concentration where half-maximal growth inhibition of tumor cells is found in comparison with a control without added inhibitor of the formula I) can lie in the range of 1 to 1000 nM, preferably from 1 to 200 nM.
The ability of the compounds of the present invention to inhibit tumor growth can be shown by the following assays with the following cell lines:
Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening
The calorimetric cytotoxicity assay used is adapted from Skehan et al (Journal of National Cancer Inst 82:1107-1112, 19901). The procedure provides a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for the National Cancer Institute""s disease-oriented in vitro anticancer-drug discovery screen.
In particular, cultures fixed with trichloroacetic acid are stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye is removed by four washes with 1% acetic acid, and protein-bound dye is extracted with 10 mM unbuffered Tris base [tris(hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results are linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm is approximately 1.5 with 1,000 cells per well.
The SRB assay provides a calorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry (Skehan et al (Journal of National Cancer Inst 82:1107-1112, 19901)).
Alternatively, the efficiency of the compounds of the formula I as inhibitors of microtubuli depolymerisation can be demonstrated as follows:
Stock solutions of the test compounds are made in DMSO and stored at xe2x88x9220xc2x0 C. Microtubuli-protein is obtained from pig brain by two cycles of temperature dependent depolymerisation/polymerisation, as described (see Weingarten et al., Biochemistry 1974; 13: 5529-37). Working stock solutions of microtubule protein (meaning tubulin plus microtubuli-associated proteins) are stored at xe2x88x9270xc2x0 C. The degree of the microtubuli protein polymerisation induced by a test compound is measured essentially as known from the literature (see Lin et al., Cancer Chem. Pharm. 1996; 38:136-140). In short, 5 xcexcl stock solution of the test compound are pre-mixed in the twenty-fold of the desired final concentration with 45 xcexcl of water at room temperature, and the mixture is then placed on ice. An aliquot of the working stock solution of pig brain microtubuli protein is thawed quickly and then diluted to 2 mg/ml with ice-cold 2xc3x97 MEM buffer (200 ml MES, 2 mM EGTA, 2 mM MgCl2, pH 6.7) (MES=2-morpholinoethanesulfonic acid, EGTA=ethylenglycol-bis-(2(2-aminoethyl)-tetraacetic acid). The polymerisation reaction is then started by the addition of each time 50 xcexcl diluted microtubuli-protein to the test compound, followed by incubation of the sample in a water bath with room temperature. Then the reaction mixtures are placed in an Eppendorf microcentrifuge and incubated for additional 15 min at room temperature. The samples are then centrifuged for 20 min at 14000 rpm at room temperature for separating polymerized from non-polymerized microtubuli protein. As indirect measure for the tubulin-polymerisation the protein concentration of the supernatant (which contains the rest of the un-polymerised, soluble microtubuti protein) is determined according to the Lowry method (DC Assay Kit, Bio-Rad Laboratories, Hercules, Calif.), and the optical density (OD) of the color reaction is determined at 750 nm with a spectrometer (SpectraMax 340, Molecular Devices, Sunnyvale, Calif.). The differences in the OD""s between samples treated with a test compound and vehicle-treated controls are compared with those of test incubations which contain 25 xcexcM Epothilon B (positive controls). The degree of polymerisation that is induced by a test compound is expressed relatively to the positive controls (100%). By comparison of several concentrations the EC50 (concentration where 50% of the maximal polymerisation is found) can be determined. For compounds of the formula I the EC50 lies preferably in the range of 1 to 200, preferably 1 to 50 xcexcM. The induction of tubulin polymerisation of test compound of the formula I in 5 xcexcM concentration as parentage in comparison to 25 xcexcM epothilone B preferably lies in the range of 50 to 100%, especially 80 to 100%.
The efficiency against tumor cells can also be shown in the following way:
Stock solutions of the test compound of formula I 10 mM) in DMSO are prepared and stored at xe2x88x9220xc2x0 C. Human KB-31 and (multidrug-resistant, P-gp 170 overexpressing) KB-8511 epidermoid carcinoma cells are from Dr. M. Baker, Roswell Park Memorial Institute (Buffalo, N.Y., USA) (for description see also Akiyama et al., Somat. Cell. Mol. Genetics 11, 117-126 (1985) und Fojo A., et al., Cancer Res. 45, 3002-3007 (1985)xe2x80x94KB-31 and KB-8511 both are derivatives of the KB-cell line (American Type Culture Collection) and are human epidermoid carcinoma cells. KB-31 cells can be cultivated in mono-layers using calf serum (M.A. Bioproducts), L-glutamine (Flow), penicillin (50 Units/ml) and streptomycin (50 xcexcg/ml (Flow); they then grow with a doubling rate of about 22 hours, and the relative efficiency of plating them out lies at about 60%. KB-8511 is a variant derived from the KB-31 cell line which has been obtained by treatment cycles with colchicine, and it shows an about 40-fold relative resistance against colchicin in comparison to KB-31 cells). The cells are incubated at 37xc2x0 C. in an incubator with 5% v/v CO2 and at 80% relative atmospheric humidity in MEM Alpha-medium which contains ribonucleosides and desoxyribonucleosides (Gibco BRL), complemented with 10 IU Penicillin, 10 xcexcg/ml Streptomycin and 5% fetal calf serum. The cells are spread in an amount of 1.5xc3x97103 cells/well in 96-well-microtiter plates and incubated overnight. Serial dilutions of the test compounds in culture medium are added at day 1. The plates are then incubated for an additional period of four days, after which the cells are fixed using 3.3% v/v glutaraldehyde washed with water and finally stained with 0.05% w/v methylen blue. After washing again, the stain is eluted with 3% HCl and the optical density at 665 nm is measured with a SpectraMax 340 (Molecular Devices, Sunnyvale, Calif.). IC50-values are determined by mathematically fitting the data to curves using the SoftPro2.0 program (Molecular Devices, Sunnyvale, Calif.) and the formula
[(OD treated)xe2x88x92(OD start)]/[(OD control)xe2x88x92(OD start)]xc3x97100.
The IC50 is defined as the concentration of a test compound at the end of the incubation period that leads to 50% of the number of cells in comparison to controls without test compound (concentration at halfmaximal inhibition of cell growth). Compounds of the formula I preferably show here and IC50 in the range from 0.1xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929 M, preferably between 0.1 and 60 nM.
Comparable testing can also be made with other tumor cell lines, such as A459 (lung; ATCC CCL 185), NCIH460 (lung), Colo 205 (colon; ATCC No. CCL 222) (HCT-15 (colon; ATCC CCL 225xe2x80x94ATCC=American Type Culture Collection (Rockville, Md., USA)), HCT-116 (colon), Du145 (prostate; ATCC No. HTB 81; see also Cancer Res. 37, 4049-58 [1978]), PC-3M (prostate-hormone-insensitive derivative obtained from Dr. I. J. Fidler (M D Anderson Cancer Center, Houston, Tex., USA) and derived from PC-3 that is a cell line available from ATCC (ATCC CRL 1435)), MCF-7 (breast; ATCC HTB 22) or MCF-7/ADR (breast, multidrug resistant; for description see Blobe G. C.et al., J. Biol. Chem. (1983), 658-664; the cell line is highly resistant (360- to 2400-fold) to doxorubicin and Vinca alkaloids compared over MDR-7 xe2x80x9cwild typexe2x80x9d cells)), where similar results are obtained as with KB-31 and KB-8511 cells. Compounds of the formula I preferably show here and IC50 in the range from 0.1xc3x9710xe2x88x929 to 500xc3x9710xe2x88x929 M, preferably between 0.1 and 60 nM.
Based on these properties, the compounds of the formula I (meaining also salts thereof) are appropriate for the treatment of proliferative diseases, such as especially tumor diseases, including also metastasis where present, for example of solid tumors, such as lung tumor, breast tumor, colorectal cancer, prostate cancer, melanoma, brain tumor, pancreas tumor, head-and-neck tumor, bladder cancer, neuroblastoma, pharyngeal tumor, or also of proliferative diseases of blood cells, such as leukaemia; or further for the treatment of other diseases that respond to treatment with microtubuli depolymerisation inhibitors, such as psoriasis. The compounds of formula I, or salts thereof, are also appropriate for covering medical implants (useful in prophylaxis of restenosis) (see WO 99/16416, priority Sep. 29, 1997).
The in vivo activity of a compound of the invention can be demonstrated with the following animal model:
Female or male BALB/c nu/nu (nude) mice are kept under sterile conditions (10 to 12 mice per Type III cage) with free access to food and water. Mice weigh between 20 and 25 grams at the time of tumor implantation. Tumors are established by subcutaneous injection of cells (minimum 2xc3x97106 cells in 100 xcexcl PBS or medium) in carrier mice (4-8 mice per cell line). The resulting tumors are serially passaged for a minimum of three consecutive transplantations prior to start of treatment. Tumor fragments (approx. 25 mg) are implanted s.c. into the left flank of animals with a 13-gauge trocar needle while the mice are exposed to Forene (Abbott, Switzerland) anesthesia.
Tumor growth and body weights are monitored once or twice weekly. All treatments are administered intravenously (i.v.) and are initiated when a mean tumor volume of approximately 100 to 250 mm3 is attained, depending upon the tumor type. Tumor volumes are determined using the formula (Lxc3x97Dxc3x97xcfx80)/6 (see Cancer Chemother. Pharmacol. 24:148-154, [1989]). Treatments with epothilones of the formula I vary the dose and the frequency of administration. Comparator agents are administered according to previously determined optimal treatment regimens. In addition to presenting changes in tu-mor volumes over the course of treatment, antitumor activity is expressed as T/C% (mean increase of tumor volumes of treated animals divided by the mean increase of tumor volu-mes of control animals multiplied by 100). Tumor regression (%) represents the smallest mean tumor volume compared to the mean tumor volume at the start of treatment, accor-ding to the formula Regression (%)=(1xe2x88x92Vend/Vstart)xc3x97100 (Vend=final mean tumor volume, Vstart=mean tumor volume at the start of treatment.
With this model, the inhibitory effect of a compound of the invention on growth e.g. of tumors derived from the following cell lines can be tested:
Human colorectal adenocarcinoma cell line HCT-15 (ATCC CCL 225) is from the American Type Culture Collection (Rockville, Md., USA), and the cells are cultivated in vitro as recommended by the supplier. HCT-15 is an epithelial-like cell line (Cancer Res. 39: 1020-25 [1979]) that is multi-drug resistant by virtue of over-expression of P-glycoprotein (P-gp, gp170, MDR-1; Anticancer Res. 11: 1309-12 [1991]; J. Biol. Chem. 264: 18031-40 [1989]; Int. J. Cancer 1991; 49: 696-703 [1991]) and glutathione-dependent resistance mechanisms (Int. J. Cancer 1991; 49: 688-95.[1991]). The Colo 205 cell line is also a human colon carcinoma cell line (ATCC No. CCL 222; see also Cancer Res. 38, 1345-55 [1978] which was isolated from ascitic fluid of a patient, dis-plays epithelial-like morphology and is generally considered to be drug-sensitive. A human androgen-independent prostate cancer cell line is used to establish subcutaneous and orthotopic models in mice. The human metastatic prostate carcinoma PC-3M is obtained from Dr. I. J. Fidler (M D Anderson Cancer Center, Houston, Tex., USA) and is cultured in Ham""s F12K media supplemented with 7% v/v FBS. The PC-3M cell line is the result of isolation from liver metastasis produced in nude mice subsequent to intrasplenic injection of PC-3 cells [ATCC CRL 1435; American Type Culture Collection (Rockville, Md., USA)], and they can grow in Eagle""s MEM supplemented with 10% fetal bovine serum, sodium pyruvate, non-essential amino acids, L-glutamine, a two-fold vitamin solution (Gibco Laboratories, Long Island, N.Y.) and penicillin-streptomycin (Flow Laboratories, Rockville, Md.). The PC-3M cell line is hormone-insensitive (that is, it grows in the absence of androgens). The PC-3 cell line is androgen receptor negative, as is presumably the derived PC-3M cell line. PC-3 is a cell line available from ATCC (ATCC CRL 1435) and corresponds to a grade IV prostatic adenocarcinoma isolated from a 62-year-old Caucasian male; the cells exhibit low acid phosphatase and testosterone-5-a-reductase activity. The cells are near-triploid with a modal number of 62 chromosomes. No normal Y chromosomes can be detected by Q-band analysis. Human lung adenocarcinoma A549 (ATCC CCL 185; isolated as explant culture from lung carcinoma tissue from a 58-year-old Caucasian male); shows epithelial morphology and can synthesize lecithin with a high percentage of desaturated fatty acids utilizing the cytidine diphosphocholine pathway; a subtelocentric marker chromosome involving chromosome 6 and the long arm of chromosome 1 is found in all metaphases. The human breast carcinoma ZR-75-1 (ATCC CRL 1500; isolated from a malignant ascitic effusion of a 63-year-old Caucasian female with infiltrating ductal carcinoma); is of mammary epithelial origin; the cells possess receptors for estrogen and other steroid hormones and have a hypertriploid chromosome number. The human epidermal (mouth) carcinoma cell line KB-8511 (a P-gp over-expressing cell line derived from the epidermoid (mouth) KB-31 carcinoma cell line) is obtained from Dr. R. M. Baker, Roswell Park Memorial Institute (Buffalo, N.Y., USA) (for description see Akiyama et al., Somat. Cell. Mol. Genetics 11, 117-126 (1985) and Fojo A., et al., Cancer Res. 45, 3002-3007 (1985)) and is cultured as previously described (Meyer, T., et al., Int. J. Cancer 43, 851-856 (1989)). KB-8511 cells, like KB-31, are derived from the KB cell line (ATCC) and they are human epidermal carcinoma cells; KB-31 cells can be grown in mono-layer using Dulbecco""s modified Eagle""s medium (D-MEM) with 10% fetal calf serum (M.A. Bioproducts), L-glutamine (Flow), penicillin (50 units/ml) and streptomycin (50 mg/ml (Flow); they then grow with a doubling time of 22 h, and their relative plating efficiency is approximately 60%. KB-8511 is a cell line derived from the KB-31 cell line by use of colchicine treatment cycles; it shows about a 40-fold relative resistance against colchicine when compared with the KB-31 cells; it can be grown under the same conditions as KB-31.xe2x80x9d
Solubility: The water solubility is determined as follows, for example: the compounds of formula I, or the salts thereof, are stirred with water at room temperature until no further compound dissolves (about 1 hour). The solubilities found are preferably between 0.01 and 1% by weight.
Within the groups of preferred compounds of formula I mentioned hereinafter, definitions of substituents from the general definitions mentioned hereinbefore may reasonably be used, for example, to replace more general definitions with more specific definitions or especially with definitions characterised as being preferred.
The invention preferably relates to a compound of the formula I wherein
R2 is absent or oxygen; xe2x80x9caxe2x80x9d can be either a single or double bond; xe2x80x9cbxe2x80x9d can be either absent or a single bond; and xe2x80x9ccxe2x80x9d can be either absent or a single bond, with the proviso that if R2 is oxygen then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are both a single bond and xe2x80x9caxe2x80x9d is a single bond; if R2 is absent then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent and xe2x80x9caxe2x80x9d is a double bond; and if xe2x80x9caxe2x80x9d is a double bond, then R2, xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent;
R3 is a radical selected from the group consisting of hydrogen; lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; and xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3;
R4 and R5 are independently selected from hydrogen, methyl or a protecting group, preferably hydrogen; and
R1 is a radical selected from the following structures: 
wherein R and Rxe2x80x2 are lower alkyl, especially methyl;
or a salt thereof where salt-forming groups are present.
The invention preferably also relates to a compound of the formula I wherein
R2 is absent or oxygen; xe2x80x9caxe2x80x9d can be either a single or double bond; xe2x80x9cbxe2x80x9d can be either absent or a single bond; and xe2x80x9ccxe2x80x9d can be either absent or a single bond, with the proviso that if R2 is oxygen then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are both a single bond and xe2x80x9caxe2x80x9d is a single bond; if R2 is absent then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent and xe2x80x9caxe2x80x9d is a double bond; and if xe2x80x9caxe2x80x9d is a double bond, then R2, xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent;
R3 is a radical selected from the group consisting of hydrogen; lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; and xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3;
R4 and R5 are independently selected from hydrogen, methyl or a protecting group, preferably hydrogen; and
R1 is a radical selected from the following structures: 
wherein Rxe2x80x2 is hydroxymethyl or fluoromethyl and R is hydrogen or methyl;
or a salt thereof where a salt-forming group is present.
The invention preferably also relates to a compound of the formula I wherein
R2 is absent or oxygen; xe2x80x9caxe2x80x9d can be either a single or double bond; xe2x80x9cbxe2x80x9d can be either absent or a single bond; and xe2x80x9ccxe2x80x9d can be either absent or a single bond, with the proviso that if R2 is oxygen then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are both a single bond and xe2x80x9caxe2x80x9d is a single bond; if R2 is absent then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent and xe2x80x9caxe2x80x9d is a double bond; and if xe2x80x9caxe2x80x9d is a double bond, then R2, xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent;
R3 is a radical selected from the group consisting of lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; and xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3,
R4 and R5 are independently selected from hydrogen, methyl or a protecting group, preferably hydrogen; and
R1 is a radical selected from the following structures: 
or a salt thereof where one or more salt-forming groups are present.
The invention preferably also relates to a compound of the formula I wherein
R2 is absent or oxygen; xe2x80x9caxe2x80x9d can be either a single or double bond; xe2x80x9cbxe2x80x9d can be either absent or a single bond; and xe2x80x9ccxe2x80x9d can be either absent or a single bond, with the proviso that if R2 is oxygen then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are both a single bond and xe2x80x9caxe2x80x9d is a single bond; if R2 is absent then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent and xe2x80x9caxe2x80x9d is a double bond; and if xe2x80x9caxe2x80x9d is a double bond, then R2, xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent;
R3 is a radical selected from the group consisting of lower alkyl other than methyl, especially ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; and xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3,
R4 and R5 are independently selected from hydrogen, methyl or a protecting group, preferably hydrogen; and
R1 is a radical of the formula 
or a salt thereof if one or more salt-forming groups are present.
The invention preferably also relates to a compound of the formula I wherein
R2 is oxygen, xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are each a single bond and xe2x80x9caxe2x80x9d is a single bond,
R3 is a radical selected from the group consisting of lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94Oxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; and xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3,
R4 and R5 are independently selected from hydrogen, methyl or a protecting group, preferably hydrogen; and
R1 is a radical selected from the group consisting of the following structures: 
or a salt thereof where one or more salt-forming groups are present.
The invention preferably also relates to a compound of the formula I wherein
R2 is absent or oxygen; xe2x80x9caxe2x80x9d can be either a single or double bond; xe2x80x9cbxe2x80x9d can be either absent or a single bond; and xe2x80x9ccxe2x80x9d can be either absent or a single bond, with the proviso that if R2 is oxygen then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are both a single bond and xe2x80x9caxe2x80x9d is a single bond; if R2 is absent then xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent and xe2x80x9caxe2x80x9d is a double bond; and if xe2x80x9caxe2x80x9d is a double bond, then R2, xe2x80x9cbxe2x80x9d and xe2x80x9ccxe2x80x9d are absent;
R3 is a radical selected from the group consisting of lower alkyl other than methyl, especially ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl or n-hexyl; xe2x80x94CHxe2x95x90CH2; xe2x80x94Cxe2x89xa1CH; xe2x80x94CH2F; xe2x80x94CH2Cl; xe2x80x94CH2xe2x80x94OH; xe2x80x94CH2xe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Oxe2x80x94CH3; and xe2x80x94CH2xe2x80x94Sxe2x80x94(C1-C6-alkyl), especially xe2x80x94CH2xe2x80x94Sxe2x80x94CH3,
R4 and R5 are independently selected from hydrogen, methyl or a protecting group, preferably hydrogen; and
R1 is a radical of the formula 
or a salt of a compound of the formula I where a salt-forming group is present.
More preferably, the invention relates to a compound of the formula Ia, 
wherein, independent of each other, the R moieties are hydrogen or methyl, or a salt thereof.
More preferably, the invention also relates to a compound of the formula Ib, 
wherein, independent of each other, the R moieties are hydrogen or methyl, or a salt thereof.
The invention most specifically also relates to a compound of the formula Ic 
wherein R* is methyl.
The invention most specifically also relates to a compound of the formula Id 
wherein A is ethyl, fluoromethyl, methoxy, methylthio or ethenyl (xe2x80x94CHxe2x95x90CH2) and D is hydrogen, fluoro, hydroxy or methyl, especially hydrogen.
The invention most specifically also relates to a compound of the formula Ie 
wherein A is ethyl, fluoromethyl, methoxy, methylthio or ethenyl (xe2x80x94CHxe2x95x90CH2) and D is hydrogen, fluoro, hydroxy or methyl.
The invention most specifically relates to the compounds of the formula I given in the examples, or the pharmaceutically acceptable salts thereof where one or more salt-forming groups are present.
Most preferably, the invention relates to a compound selected from the group consisting of compound D (Example 1), the compound of Example 2 A), the compound of Example 2C, compound 18b (see Example 4), compound 19b (see Example 4), compound 46 (see Example 4), compound 50 (see Example 4), compound 52 (see Example 4), compound 53 (see Example 4), compound 58 (see Example 4), compound 59 (see Example 4), compound 66 (see Example 4), compound 67 (see Example 4), and compound 68 (see Example 4), or a pharmaceutically acceptable salt thereof if one or more salt-forming groups are present.
The compounds of the invention can be synthesized using methods in analogy to methods that are known in the art, preferably by a method characterized by
a) reacting a iodide of the formula II, 
wherein R2, R3, R4, R5, a, b and c and the waved bond have the meanings given under formula I, with a tin compound of the formula III,
R1xe2x80x94Sn(R)3xe2x80x83xe2x80x83(III)
wherein R1 has the meanings given under formula I and R is lower alkyl, especially methyl or n-butyl, or
b) reacting a tin compound of the formula IV, 
wherein R2, R3, R4, R5, a, b and c and the waved bond have the meanings given under formula I, with a iodide of the formula V,
R1xe2x80x94Ixe2x80x83xe2x80x83(V)
wherein R1 has the meanings given under formula I;
and, if desired, a resulting compound of the formula I is converted into a different compound of the formula I, a resulting free compound of the formula I is converted into a salt of a compound of the formula I, and/or a resulting salt of a compound of the formula I is converted into a free compound of the formula I or into a different salt of a compound of the formula I, and/or a stereoisomeric mixture of compounds of formula I is separated into the corresponding isomers.
In all starting materials, where required, functional groups that shall not participate in the reaction are protected by protecting groups, especially standard protecting groups. The protecting groups, their introduction and their cleavage are known in the art, for example, they are described in the standard references mentioned above.
Reaction a): The reaction (a (preferably improved) Stille coupling) preferably takes place under standard conditions; more preferably, the reaction takes place
(i) in an appropriate solvent, e.g. toluene, at elevated temperature, especially about 90 to about 100xc2x0 C., preferably with an excess of the tin compound of the formula III, preferably in the 1.1- to 3-, e.g. the 1.5- to 2-fold molar excess; and a catalytic amount, preferably of about 1 to 30%, preferably 5 to 10%, of Pd(PPh3)4; or
(ii) in an appropriate solvent, e.g. dimethylformamide (DMF), at temperatures of from 10 to 40xc2x0 C., especially at 25xc2x0 C., preferably with an excess of the tin compound of the formula III, preferably in the 1.1- to 3-, e.g. the 1.5- to 2.3-fold molar excess; in the presence of a catalytic amount, preferably of 10 to 50%, especially 20 to 30%, of Pd(MeCN)2Cl2. Alternative conditions for this coupling also comprise the use of the following reagents and/or conditions:
(iii). cuprous 2-thiophene carboxylate, N-methyl-2-pyrrolidine.
(iv). PdCl2(MeCN)2 (cat.), DMF, 50-150xc2x0 (with or without additon of tertiary base).
(v). Pd(PPh3)4/Cul (cat), DMF, 50-150xc2x0 (with or without addition of tertiary base)
Reaction b): The reaction (an improved Stille coupling) preferably takes place under standard conditions; more preferably, the reaction takes place in an appropriate solvent, especially DMF, at temperatures of from 50 to 100, preferably from 80 to 85xc2x0 C., preferably with an excess of the iodide of the formula V, in the presence of a catalytic amount of AsPh3, preferably about 0.4 equivalents, Cul, preferably about 0.1 equivalents, and PdCl2(MeCN)2, preferably about 0.2 equivalents.
Especially preferred are the reaction conditions mentioned in the examples.
Conversions of Compounds/salts
Compounds of the formula I can be converted into different compounds of formula I by standard or novel methods.
For example, a compound of the formula I wherein R2 is absent, b and c are absent and a is a double bond, and the other moieties are as described for compounds of the formula I, can be converted into the corresponding epoxide wherein R2 is O and b and c are present while a is a single bond. Preferably, the epoxidation takes place in the presence of (+)-diethyl-D-tartrate ((+)-DET) (preferably about 0.5 equivalents), Ti(i-PrO)4 (preferably about 0.5 equivalents), tert-butylhydroperoxide (preferably about 2.2 equivalents) and molecular sieve, especially 4 xc3x85 molecular sieves, in an appropriate solvent, e.g. methylene chloride and optionally an alkane, such as decane, at low temperatures, preferably -78 to 0xc2x0 C., especially about xe2x88x9240xc2x0 C.; or in presence of hydrogen peroxide (preferably about 30 equivalents), acetonitrile (preferably about 60 equivalents), a base, especially KHCO3 (preferably about 10 equivalents) in an appropriate solvent, e.g. an alcohol, preferably methanol, at preferred temperatures between 10 and 40xc2x0 C., e.g. at about 25xc2x0 C.
A compound of the formula I wherein R3 is hydroxymethyl can be converted into a compound of formula I wherein R3 is fluoromethyl, e.g. by treatment with DAST (preferably 1.05 to 1.4 equivalents) in an appropriate solvent, e.g. methylene chloride, at low temperatures, preferably at xe2x88x9295 to 0xc2x0 C., especially at about xe2x88x9278xc2x0 C. DAST is diethylamino-sulfur trifluoride.
A compound of the formula I wherein R3 is iodomethyl can be converted into a compound of formula I wherein R3 is methyl, e.g. by treatment with cyanoborohydride (preferably about 10 equivalents) in HMPA (hexamethylphosphoric triamide) at elevated temperatures, e.g. at 40 to 45xc2x0 C.
Other conversions can be made in accordance with known procedures, e.g. those given in PCT application WO 98/25929, which is herewith incorporated by reference.
Salts of a compound of formula I with a salt-forming group may be prepared in a manner known per se. Acid addition salts of compounds of formula I may thus be obtained by treatment with an acid or with a suitable anion exchange reagent.
Salts can usually be converted to free compounds, e.g. by treating with suitable basic agents, for example with alkali metal carbonates, alkali metal hydrogencarbonates, or alkali metal hydroxides, typically potassium carbonate or sodium hydroxide.
The resulting free compounds can then, if desired, be converted into different salts as described for the formation of salts from the free compounds.
Stereoisomeric mixtures, e.g. mixtures of diastereomers, can be separated into their corresponding isomers in a manner known per se by means of suitable separation methods. Diastereomeric mixtures for example may be separated into their individual diastereomers by means of fractionated crystallization, chromatography, solvent distribution, and similar procedures. This separation may take place either at the level of a starting compound or in a compound of formula I itself. Enantiomers may be separated through the formation of diastereomeric salts, for example by salt formation with an enantiomer-pure chiral acid, or by means of chromatography, for example by HPLC, using chromatographic substrates with chiral ligands.
Starting Materials
Starting materials and intermediates are known in the art, commercially available, and/or prepared in accordance with methods known in the art or in analogy thereto.
Compounds of the formula II and of the formula III can, for example, be synthesized as described in PCT application WO 98/25929, which is herewith incorporated by reference, or as described or in analogy to the methods in the examples.
Compounds of the formula IV are accessible by reaction of the respective compounds of the formula II, for example by reaction of a compound of the formula II with (R)6Sn2, wherein R is lower alkyl, especially methyl or n-butyl, in the presence of an appropriate nitrogen base, e.g. Hxc3xcnig""s base, and in the presence of catalytic amount (preferably about 0.1 equivalents) of Pd(PPh3)4 in an appropriate solvent, e.g. toluene, at elevated temperatures, e.g. 30 to 90xc2x0 C., especially 80 to 85xc2x0 C. lodides of the formula V are known and can be obtained according to literature procedures, or they are commercially available. For example, 2-iodo-6-methyl pyridine can be obtained according to Klei, E.; Teuben, J. H. J. Organomet. Chem. 1981, 214, 53-64; 2-iodo-5-methyl pyridine according to Talik, T.; Talik, Z. Rocz. Chem. 1968, 42, 2061-76; and 2-iodo-4-methyl pyridine according to Talik, T.; Talik, Z. Rocz. Chem. 1968, 42, 2061-76, Yamamoto, Y.; Yanagi, A. Heterocycles 1981, 16, 1161-4 or Katritzky, A. R.; Eweiss, N. F.; Nie, P.-L. JCS, Perkin Trans I 1979, 433-5. The corresponding hydroxymethyl-substituted compounds of formula V are available for example by oxidation of the methyl groups of the iodides mentioned above with SeO2 and subsequent reduction, e.g. with NaBH4 or DIBALH) of the aldehyde or by oxidation of the methyl group to form the acid (for example with KMnO4) and subsequent reduction of the ester e.g. with DIBAL.
Preferably, new or also known starting materials and intermediates can be prepared in accordance with or in analogy to the methods described in the examples, where the amounts, temperatures and the like of the respective reactions can be modified, e.g. by varying in the range of xc2x199%, preferably xc2x125%, and other appropriate solvents and reagents can be used.
The invention relates also to all new intermediates, especially those mentioned in the Examples.
The invention also relates to a method of synthesis of a compound of the formula VI 
which is characterized in that a compound of the formula VII 
wherein R3 is lower alkyl, especially methyl or n-butyl, is coupled with a iodide of the formula VIII, 
(commercially available, e.g. from TCI, USA), especially under Stille coupling and analogous/modified conditions; especially in an appropriate solvent, especially a di-lower alkyl-lower alkanoyl amide, preferably dimethyl formamide or -acetamide; the compound of formula VIII preferably being in slight molar excess over the compound of the formula VII, e.g. in the 1.1- to 5-fold, especially in the 1.5 to 2.5-fold excess, for example in 2.1-fold excess; in the presence of catalytic amounts of AsPh3 (especially about 0.4 equivalents), PdCl2(MeCN)2 (especially about 0.2 equivalents) and Cul (especially about 0.1 equivalents); at elevated temperatures, e.g. in the range of 50 to 90xc2x0 C., preferably about 80 to about 85xc2x0 C. For further reaction conditions see the detailed description under process variant (a) (xe2x80x9cReaction a)xe2x80x9d) above for the synthesis of a compound of the formula I. The reaction conditions can be optimized for the particular substrates in accordance with the know-how of the person havin skill in the art.
The invention also relates to the inverted method wherein instead of the compound of the formula VII an analogue is used where instead of the Sn(R)3 moiety a iodine is present and instead of the compound of the formula VIII an analogue is used that has the moiety Sn(R)3 instead of the iodine. The reaction conditions are then analogous to those under method a) presented above for the synthesis of a compound of the formula I.
The invention also relates to a method of synthesis for epothilones E and especially F of the formula IX, 
wherein Q is hydrogen (epothilone E) or methyl (epothilone F), which is characterized in that a compound of the formula X 
is epoxidized in the presence of a peroxide to the compound of the formula IX, preferably by using standard reaction conditions for epoxidation, more preferably by epoxidation in the presence of H2O2, a base, especially KHCO3, acetonitrile and an appropriate solvent, especially an alcohol, e.g. methanol, at temperatures preferably in the range of 0 to 50xc2x0 C., especially about 25xc2x0 C. (in-situ-formation if methylperoxycarboximidic acid); or in the presence of (+)-diethyl-D-tartrate and titanium isopropoxide, then t-butyl hydroperoxide in an appropriate solvent, e.g. methylenchloride and optionally decane at low temperatures, e.g. xe2x88x9278 to 0xc2x0 C., especially about xe2x88x9240xc2x0 C.
These reactions have inter alia the advantage to provide the final products in high yield and good isomeric purity.
Pharmaceutical Preparations
The present invention also relates to the use of a compound of the formula I for the manufacture of a pharmaceutical formulation for use against a proliferative disease as defined above; or to a pharmaceutical formulation for the treatment of said proliferative disease comprising a compound of the invention and a pharmaceutically acceptable carrier.
The compounds of the formula I are called active ingredient hereinafter.
The invention relates also to pharmaceutical compositions comprising an active ingredient as defined above, for the treatment of a proliferative disease, especially as defined above, and to the preparation of pharmaceutical preparations for said treatment.
The invention relates also to a pharmaceutical composition that is suitable for administration to a warm-blooded animal, especially a human, for the treatment of a proliferative disease as defined hereinbefore, comprising an amount of an active ingredient, which is effective for the treatment of said proliferative disease, together with at least one pharmaceutically acceptable carrier. The pharmaceutical compositions according to the invention are those for enteral, such as nasal, rectal or oral, or preferably parenteral, such as intramuscular or intravenous, administration to a warm-blooded animal (human or animal), that comprise an effective dose of the pharmacologically active ingredient, alone or together with a significant amount of a pharmaceutically acceptable carrier. The dose of the active ingredient depends on the species of warm-blooded animal, the body weight, the age and the individual condition, individual pharmacokinetic data, the disease to be treated and the mode of administration; preferably, the dose is one of the preferred doses as defined below, being accommodated appropriately where pediatric treatment is intended.
The pharmaceutical compositions comprise from about 0.00002 to about 95%, especially (e.g. in the case of infusion dilutions that are ready for use) of 0.0001 to 0.02%, or (for example in case of infusion concentrates) from about 0.1% to about 95%, preferably from about 20% to about 90%, active ingredient (weight by weight, in each case). Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, dragees, tablets or capsules.
The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes.
Solutions of the active ingredient, and also suspensions, and especially isotonic aqueous solutions or suspensions, are preferably used, it being possible, for example in the case of lyophilized compositions that comprise the active ingredient alone or together with a pharmaceutically acceptable carrier, for example mannitol, for such solutions or suspensions to be produced prior to use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known per se, for example by means of conventional dissolving or lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing substances, such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.
Suspensions in oil comprise as the oil component the vegetable, synthetic or semi-synthetic oils customary for injection purposes. There may be mentioned as such especially liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brasidic acid or linoleic acid, if desired with the addition of anti-oxidants, for example vitamin E, betacarotene or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of those fatty acid esters has a maximum of 6 carbon atoms and is a mono- or polyhydroxy, for example a mono-, di- or tri-hydroxy, alcohol, for example methanol, ethanol, propanol, butanol or pentanol or the isomers thereof, but especially glycol and glycerol.
The injection or infusion compositions are prepared in customary manner under sterile conditions; the same applies also to introducing the compositions into ampoules or vials and sealing the containers.
Preferred is an infusion formulation comprising an active ingredient and a pharmaceutically acceptable organic solvent.
The pharmaceutically acceptable organic solvent used in a formulation according to the invention may be chosen from any such organic solvent known in the art. Preferably the solvent is selected from alcohol, e.g. absolute ethanol or ethanol/water mixtures, more preferably 70% ethanol, polyethylene glycol 300, polyethylene glycol 400, polypropylene glycol or N-methylpyrrolidone, most preferably polypropylene glycol or 70% ethanol or polyethylene glycol 300.
The active ingredient may preferably be present in the formulation in a concentration of about 0.01 to about 100 mg/ml, more preferably about 0.1 to about 100 mg/ml, still more preferably about 1 to about 10 mg/ml (especially in infusion concentrates).
The active ingredient may be used as pure substances or as a mixture with another active ingredient. When used in its pure form it is preferable to employ a concentration of active ingredient of 0.01 to 100, more preferably 0.05 to 50, still more preferably 1 to 10 mg/ml (this number makes reference especially to an infusion concentrate that, before treatment, is diluted accordingly, see below).
Such formulations are conveniently stored in vials or ampoules. Typically the vials or ampoules are made from glass, e.g. borosilicate or soda-lime glass. The vials or ampoules may be of any volume conventional in the art, preferably they are of a size sufficient to accommodate 0.5 to 5 ml of formulation. The formulation is stable for periods of storage of up to 12 to 24 months at temperatures of at least 2 to 8xc2x0 C.
Formulations must be diluted in an aqueous medium suitable for intravenous administration before the formulation of the active ingredient can be administered to a patient.
The infusion solution preferably must have the same or essentially the same osmotic pressure as body fluid. Accordingly, the aqueous medium preferably contains an isotonic agent which has the effect of rendering the osmotic pressure of the infusion solution the same or essentially the same as body fluid.
The isotonic agent may be selected from any of those known in the art, e.g. mannitol, dextrose, glucose and sodium chloride. Preferably the isotonic agent is glucose or sodium chloride. The isotonic agents may be used in amounts which impart to the infusion solution the same or essentially the same osmotic pressure as body fluid. The precise quantities needed can be determined by routine experimentation and will depend upon the composition of the infusion solution and the nature of the isotonic agent. Selection of a particular isotonic agent is made having regard to the properties of the active agent.
The concentration of isotonic agent in the aqueous medium will depend upon the nature of the particular isotonic agent used. When glucose is used it is preferably used in a concentration of from 1 to 5% w/v, more particularly 5% w/v. When the isotonic agent is sodium chloride it is preferably employed in amounts of up to 1% w/v, in particular 0.9% w/v.
The infusion formulation may be diluted with the aqueous medium. The amount of aqueous medium employed as a diluent is chosen according to the desired concentration of active ingredient in the infusion solution. Preferably the infusion solution is made by mixing a vial or ampoule of infusion concentrate afore-mentioned with an aqueous medium, making the volume up to between 20 ml and 200 ml, preferably between about 50 and about 100 ml, with the aqueous medium.
Infusion solutions may contain other excipients commonly employed in formulations to be administered intravenously. Excipients include antioxidants. Infusion solutions may be prepared by mixing an ampoule or vial of the formulation with the aqueous medium, e.g. a 5% w/v glucose solution in WFI or especially 0.9% sodium chloride solution in a suitable container, e.g. an infusion bag or bottle. The infusion solution, once formed, is preferably used immediately or within a short time of being formed, e.g. within 6 hours. Containers for holding the infusion solutions may be chosen from any conventional container which is nonreactive with the infusion solution. Glass containers made from those glass types afore-mentioned are suitable although it may be preferred to use plastics containers, e.g. plastics infusion bags.
The invention also relates to a method of treatment of a warm-blooded animal, especially a human, that is in need of such treatment, especially of treatment of a proliferative disease, comprising administering a compound of the formula I, or a pharmaceutically acceptable salt thereof, to said warm-blooded animal, especially a human, in an amount that is sufficient for said treatment, especially effective against said proliferative disease.
Dosage forms may be conveniently administered intravenously in a dosage of from 0.01 mg up to 100 mg/m2 of active ingredient, preferably from 0.1 to 20 mg/m2 of active ingredient. The exact dosage required and the duration of administration will depend upon the seriousness of the condition, the condition of the patient and the rate of administration. The dose may be administered daily or preferably with intervals of some days or weeks, for example weekly or every 3 weeks. As the dose may be delivered intravenously, the dose received and the blood concentration can be determined accurately on the basis of known in vivo and in vitro techniques.
Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragee cores or capsules. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.
The compounds of the invention can be used alone or in combination with other pharmaceutically active substances, e.g. with other chemotherapeutics, such as classical cytostatics. In the case of combinations with an other chemotherapeutic, a fixed combination of two or more components or two or more independent formulations (e.g. in a kit of part) are prepared as described above, or the other chemotherapeutics are used in standard formulations that are marketed and known to the person of skill in the art, and the compound of the present invention and any other chemotherapeutic are administered at an interval that allows for a common, additional or preferably synergistic effect for tumor treatment.